1. Field of the Invention
The present invention relates to the field of serum preparations for use in the in vitro production of IgG antibodies, and to methods for preparing serum depleted of intrinsic immunoglobulin. The invention also relates to the field of antibody preparation, including improved methods for the production of monoclonal antibodies in cell culture media.
2. Background of the Related Art
The use of pure monoclonal antibodies (MAb) both as therapeutic reagents in clinical practice and as immunological agents in biomedical research is growing steadily. Monoclonal antibodies are produced as an animal ascites fluid or from cell culture supernatants. The production of MAb in vitro by hybridoma cells, B lymphoblasts, Ig transfectomas and myeloma cell lines has many advantages over the use of animal ascites, which may contain both known and unknown viruses, natural antibodies, and other potentially hazardous substances.
These antibody producing cells (Ab-cell lines), typically grow better in vitro in medium supplemented with whole animal serum. In fact, the majority of B cell hybridomas grown in tissue culture medium require or prefer the presence of added whole animal sera. Whole animal serum, i.e., serum that has not been processed to exclude immunoglobulins and/or other naturally occurring substances contains many as yet unidentified constituents that appear to be necessary for optimal cell growth.
One of the many drawbacks of in vitro growth of Ab-cell lines in cell culture media with whole serum is the high cost of isolating and purifying antibody from the spent culture media. For example, whole serum introduces a relatively large concentration of immunoglobulins, such as IgG, to the culture system. Therefore, IgG antibody preparations from spent media of IgG MAb producing cultures is relatively impure, as the majority of the IgG in such media is derived from the serum source, and not the Ab-cell line.
Conventionally, the method of production of MAb has been to grow the Ab-cell line in serum which contains serum derived antibodies. The spent serum is then contacted with protein G or protein A which separates the IgG antibodies in the medium from the other medium components. These IgG antibodies are then eluted from the protein G or protein A. However, the antibodies so derived are a mixture of Ab-cell line produced IgG and the serum derived IgG. The desired IgG antibodies are then separated from the serum derived IgG, usually by an affinity chromatography column that is specific for the desired IgG.
These procedures are laborious and can handle only relatively small volumes of media containing antibody. In addition, the preparation of commercially valuable quantities of monoclonal antibody requires the processing of large volumes of tissue culture supernatant because the concentration of MAb is low and the antibody represents only a small fraction of the immunoglobulin present (Jiskoot et al., 1991).
To bypass and simplify the media purification procedure, Ab-cell lines may also be cultured in media that does not include serum at all, or that includes a serum substitute. A number of serum substitutes have been developed (Barnes, overview, 1987; Schneider, 1989; Federspiel et al., 1991), some of which are commercially available (Mariani et al., 1991). .gamma.-Globulins Reduced Newborn Calf Serum (Sigma, USA) and Nu-Serum (Collaborative Research, USA) are two such available serum substitutes. However, the .gamma.-Globulins Reduced Newborn Calf Serum (Sigma, USA) was found by the present inventors to be incapable of maintaining the growth of certain Ab-cell lines, such as TVE-1 cells. It is contemplated by the present inventors that the Cohn fractionation process, in which ethanol is used to precipitate .gamma.-globulins, destroys constituents of the serum that are important in the growth of TVE-1 cells, and potentially other cell lines. In addition, this particular serum substitute was found by the present inventors to contain unacceptable amounts of bovine IgG.
Nu-Serum (Collaborative Research, USA) is a second exemplary serum substitute, and includes a reduced amount of IgG. However, this preparation has been reported to contain 2.5% serum (Mariani et al., 1991), and has been found to contain significant amounts of bovine IgG.
Studies by the present inventors also demonstrate that TVE-1 cell growth in culture media with the Nu-Serum preparation is inferior to cell growth in non-IgG depleted FCS-containing media. TVE-1 cell growth and antibody production in media containing Nu-Serum has also been observed by the present inventors to be significantly inferior to that achieved in media containing the IgG depleted serum of the present invention.
An additional disadvantage of currently available serum-free medias and serum substitutes, such as the Nu-Serum and others described herein, is that they frequently require time consuming modification for use with particular cell lines (Glassy et al., review, 1988). Thus, media that contain serum remain the standard, and are currently preferred for the growth of most cell lines and hybridomas (Jayme and Blackman, 1985).
Currently available modified serum culture supplements and serum-free medias fail to meet existing needs in the industry for optimal growth of Ab-cell lines and cost effective production and isolation of commercially significant amounts of antibody from cell culture. While serum-free or serum modified media may offer some advantages (Barnes, 1987), their use has the disadvantages of reduced cell growth and the necessity for costly and time consuming purification techniques. Therefore, there still exists an immediate need for a media supplement that supports cell growth comparable to that observed in serum-containing media, without the necessity of extensive costly and time consuming purification of the IgG.